DESCRIPTION(Provided by applicant): NMDA receptors are glutamate-gated ion channels that mediate the slow component of fast excitatory synapses.Intrinsic channel activation properties shape the post-synaptic current and contribute to critical physiological |processes in neurodevelopment and synaptic plasticity. The kinetic mechanism of NMDA receptor activation iscomplex and poorly understood. However, it represents the molecular basis of glutamate excitotoxicity underlyingneuronal cell death that occurs in stroke, epilepsy and neurodegenerative disorders. We will develop a kineticstate model describing the NMDA receptor activation and will use this model to identify structure -functionrelationships in the glutamate-binding domain. In Aim 1, single-channel currents elicited by steady-stateconcentrations of glutamate will be recorded in cell-attached patches from HEK 293 cells that express recombinantNR1/NR2A channels. We will use kinetic modeling to determine the number of closed and open states, theinterconnections between these states, and the corresponding rate constants that best describe these data. InAim 2, we will test and refine the model developed in Aim1 by using it to fit currents recorded from NR1/NR2Areceptors in excised patches, during jumps into different concentrations of glutamate. In Aim 3, we will recordsingle channel currents from NMDA receptors carrying single-substitution mutations in NR2A and NR2B subunitsand calculate rate constants for the microscopic transitions postulated by our kinetic model. This analysis willprobe the mechanism of glutamate binding, channel gating and/or desensitization. A description of the NMDAreceptor activation kinetics and these structure-function relationships will enhance our understanding of fastexcitatory synapses and will become instrumental in developing specifically targeted therapies.